Growth Hormone ("GH") is a peptide having 191 amino acids which stimulates the production of numerous different growth factors, e.g. IGF-I and so promotes growth of numerous tissues (skeleton, connective tissue, muscle and viscera) and physiological activities (raising nucleic acid and protein synthesis and lipolysis, but lowering urea secretion).
Release of GH is under the control of releasing and inhibiting factors secreted by the hypothalamus. The primary releasing factor is growth hormone releasing hormone ("GH-RH"); human growth hormone-releasing hormone ("hGH-RH") is a peptide having 44 amino acids. The novel peptides of the present invention relate to analogues of hGH-RH having only residues 1 through 29 ("hGH-RH(1-29)NH.sub.2 "), i.e., to analogues of the peptide which has the amino acid sequence:
Tyr-Ala-Asp-Ala-Ile.sup.5 -Phe-Thr-Asn-Ser-Tyr.sup.10 -Arg-Lys-Val-Leu-Gly.sup.15 -Gln-Leu-Ser-Ala-Arg.sup.20 -Lys-Leu-Leu-Gln-Asp.sup.25 -Ile-Met-Ser-Arg.sup.29 -NH.sub.2 PA1 R.sup.1 -R.sup.2 -Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-L ys-Leu-Leu-Gln-Asp-Ile-R.sup.27 -Ser-Arg-NH.sub.2, PA1 R.sup.1,R.sup.2,Ala.sup.8,R.sup.15,Nle.sup.27 !hGH-RH(1-29)-NH.sub.2, PA1 wherein X is H, Ac, Aqc, IAc, BrProp, For, Ibu, Nac, 2-Nac, 1- or 2-Npt, 1- or 2-Npr, PhAc, Fpr, or any other aromatic or nonpolar acyl group, PA1 R.sup.1 is Tyr, His or Phe(Y), in which Y=H, F, Cl, Br, NO.sub.2, NH.sub.2, CH.sub.3 or OCH.sub.3, PA1 R.sup.2 is D-Arg, D-Cit, D-Har, D-Lys, D-Tic or D-Orn, PA1 R.sup.3 is Asp, D-Asp, Ala, D-Ala or Gly, PA1 R.sup.4 is Ala, Abu or Gly, PA1 R.sup.5 is Ile, Ala or Gly, PA1 R.sup.6 is Phe, Tic, Ala, Pro, Tpi, Nal or Phe(Y), in which Y=H, F, Cl, Br, NO.sub.2, NH.sub.2, CH.sub.3 or OCH.sub.3, PA1 R.sup.8 is Asn, Gin, Ser, Thr, Val, Leu, lie, Ala, D-Ala, D-Asn, D-Gln, D-Thr, D-Leu, Abu, D-Abu, Nle, or Aib, PA1 R.sup.9 is Ser PA1 R.sup.10 is Tyr or Phe(Y), in which Y=H, F, Cl, Br, NO.sub.2, CH.sub.3 or OCH.sub.3, PA1 R.sup.11 is Arg, D-Arg, or Cit, PA1 R.sup.12 is Lys, D-Lys, Cit, D-Cit, Orn, D-Orn, NIe or Ala, PA1 R.sup.15 is Gly, Ala, Abu or GIn, PA1 R.sup.19 is Ala or Abu, PA1 R.sup.20 is Arg, D-Arg or Cit, PA1 R.sup.21 is Lys, D-Lys, Orn or Cit, PA1 R.sup.27 is Met, Nle or Abu, PA1 R.sup.28 is Ser, Asn, Asp, Ala or Abu, PA1 R.sup.29 is Agm, Arg-NH.sub.2, Arg-OH, Cit-NH.sub.2, Cit-OH, Har-NH.sub.2 or Har-OH, and pharmaceutically acceptable salts thereof. PA1 X is H, Ac, Aqc, IAc, BrProp, For, Ibu, Nac, 2-Nac, 1- or 2-Npt, 1- or 2-Npr, PhAc, Fpr or any other aromatic or nonpolar acyl group, PA1 A is Glu, D-Glu, Gin, Asp, D-Asp, Asn, Abu, Leu, Tyr, His, Phe(Y), in which Y=H, F, Cl, Br, NO.sub.2, NH.sub.2, CH.sub.3 or OCH.sub.3, Ser, Thr, Val, lie, Ala, D-Ala, D-Asn, D-Gin, D-Thr, D-Leu, Abu, D-Abu, Nle, or Aib, PA1 B is Lys, Lys-OH, Lys-NH.sub.2, D-Lys, D-Lys-OH, D-Lys-NH.sub.2, Arg, Arg-OH, Arg-NH.sub.2, D-Arg, D-Arg-OH, D-Arg-NH.sub.2, Orn, Orn-OH, Orn-NH.sub.2, D-Orn, D-Orn-OH, D-Orn-NH.sub.2, Har, Har-OH, Har-NH.sub.2, or Agm PA1 R.sup.4 is Ala, Abu or Gly, PA1 R.sup.5 is Ile, Ala or Gly, PA1 R.sup.6 is Phe, Tic, Tpi, Nal or Phe(Y), in which Y=H, F, Cl, Br, NO.sub.2, NH.sub.2, CH.sub.3 or OCH.sub.3, PA1 R.sup.10 is Tyr or Phe(Y), in which Y=H, F, Cl, Br, NO.sub.2, NH.sub.2, CH.sub.3 or OCH.sub.3, PA1 R.sup.15 is Gly, Ala, Abu or GIn, PA1 R.sup.27 is Met, Nle or Abu, PA1 R.sup.28 is Ser, Asn, Asp, Ala or Abu,
GH has been implicated in several diseases. One disease in which GH is involved is acromegaly, in which excessive levels of GH are present. The abnormally enlarged facial and extremity bones of this disease can be treated by administering a GH-RH antagonist.
Further diseases involving GH are diabetic retinopathy and diabetic nephropathy. The damage to the retina and kidneys respectively in these diseases, believed to be due to GH, results in blindness or reduction in kidney function. This damage however can be prevented or slowed by administration of an effective GH-RH antagonist.
In an effort to intervene in these disease and other conditions, some investigators have attempted to control GH levels by using somatostatin, one inhibitor of GH release. However, somatostatin, if administered alone, does not suppress GH or IGF-I levels to a desired degree. If administered in combination with a GH-RH antagonist, somatostatin would improve suppression of IGF-I levels much better.
Other workers have investigated various modifications of GH-RH to elucidate the relationship of the structure of GH-RH to its activity in an effort to provide synthetic congeners with improved agonistic or antagonistic properties. (Synthesis may be by solid phase method, described in U.S. Pat. No. 4,914,189, or in liquid phase, as described in U.S. Pat. No. 4,707,541.) Thus, in one study, it was found that synthesizing GH-RH without its N-terminus residue--i.e., forming hGH-RH(2-44)--results in an analogue having GH releasing activity which is only 0.1% that of GH-RH. By contrast, synthesizing a GH-RH analogue without its residues 30 through 44--i.e., synthesizing hGH-RH(1-29)NH.sub.2 --results in an analogue which retains 50% or more of the potency of native hGH-RH. Synthesizing even shorter analogues--e.g., GH-RH(1-28)NH.sub.2 or GH-RH(1-27)NH.sub.2 --resulted in substantially lower bioactivity. These results indicate that sequence 1-29 is important to the bioactivity of GH-RH.
In another study, it was found that acetylating the N-terminus amino acid residue of GH-RH or replacing it with a D-isomer--thus forming Ac-Tyr.sup.1 !GH-RH or D-Tyr.sup.1 !GH-RH--lowers the ability of the analogues to release GH to 2-3% that of GH-RH. These analogues also have less affinity in vitro for GH-RH binding sites. By contrast, acetylation of the alpha amino group of residue 1 in hGH-RH(1-29)NH.sub.2 --thus forming AcTyr.sup.1 !hGH-RH(1-29)NH.sub.2 --is found to raise the in vivo potency over that of GH-RH by ten fold or more.
In further studies, it was found that Ac-Tyr.sup.1,D-Arg.sup.2 !hGH-RH(1-29) NH.sub.2 antagonizes the activation of rat anterior pituitary adenylate cyclase by hGH-RH(1-29)NH.sub.2. The same peptide was found to block the action of GH-RH on its receptors in the pituitary and hypothalamus, and to inhibit the pulsatile growth hormone secretion.
Several reported modifications to GH-RH have resulted in agonistic activity. U.S. Pat. No. 4,659,693 discloses agonists of hGH-RH(1-29) having the formula:
wherein R.sup.1 is H, Tyr or His; R.sup.2 may be various residues; and R.sup.27 is Nle. These agonists are said to stimulate release of growth hormone releasing factor ("GRF") and so to be suitable in pharmaceutical compositions. ("GRF" is merely a synonym for GH-RH, and the latter abbreviation is used hereinafter, despite use of GRF in U.S. Pat. No. 4,659,693 and other publications.)
U.S. Pat. No. 4,914,189 discloses other analogues of GH-RH which are agonists. In these agonists, the N-terminus group Q.sup.1 CO--, where Q.sup.1 signifies certain omega or alpha-omega substituted alkyl groups, may be Tyr or des-amino-Tyr; the C-terminus group NH-Q.sup.2, where Q.sup.2 signifies certain lower omega-guanidino-alkyl groups, may be Agm; and R.sup.27 may be Nle. These analogues are said to be extremely potent stimulants of GH release and to enjoy high resistance to in vivo enzymatic degradation due to the omega-guanidino-lower alkyl group at the C-terminus.
Published application WO 91/16923 reviews earlier attempts to alter the secondary structure of hGH-RH by modifying its amino acid sequence. These earlier attempts include: replacing Tyr.sup.1, Ala.sup.2, ASp.sup.3 or Asn.sup.8 with their D-isomers; replacing Ser.sup.9 with Ala to enhance amphilicity of the region; and replacing Asn.sup.8 with L- or D-Ser, D-Arg, Asn, Thr, Gln or D-Lys. Certain of these modifications are said to enhance GH releasing activity. WO 91/16923 also states that replacing Asn.sup.8 with Ala induces an enormous increase in GH releasing activity. The peptides said to have this benefit have the formula:
where R.sup.1 is Dat or A-R.sup.1, where A is lower acyl or benzyl and R.sup.1 includes Tyr and His; R.sup.2 is Ala, D-Ala or N-Me-D-Ala (N-Methyl-D-Ala); and R.sup.15 may include Gly, Ala or Aib. One preferred embodiment has R.sup.8,9,15 as Ala. It is noted that R.sup.8 in this publication is never Asn. Pharmaceutical compositions for enhancing growth are further disclosed.
European Patent Application Ser. No. 0 413 839 A, filed Aug. 22, 1989, assigned to the same assignee as the present application, discloses analogues of hGH-RH(1-29)-NH.sub.2 said to have enhanced release of GH. The 29 as follows: R.sup.1 may be Tyr or Dat; R.sup.2 may be L or D Ala; R.sup.8 may be Asn or Ser; R.sup.12 may be L or D isomers of Lys, Arg or Orn; R.sup.15 may be Gly or Ala; R.sup.27 may be NIe; R.sup.28 may be Asp, Asn or Ser; and R.sup.29 may be Agm. However, residue 6 is never replaced: it is always Phe.
Yet another modification of hGH-RH was disclosed in U.S. Pat. No. 5,183,660, where GH-RH was conjugated with polyethylene glycol derivatives. The resulting conjugate was said to exhibit decreased antigenicity, delay in biological clearance in vivo and physiological activity over a longer time.
In several of these investigations, it was found that variants of the hGH-RH agonistic analogues had antagonistic, rather than agonistic, activity. Thus, in U.S. Pat. No. 4,659,693 (where R.sup.2 may be certain D-Arg residues substituted with alkyl groups), when R.sup.1 is H, the hGH-RH analogues are said to act as antagonists. Similarly, in WO 91/16923, discussed above, if R.sup.2 in the analogues is D-Arg, and R.sup.8, R.sup.9, and R.sup.15 are substituted as indicated above, antagonistic activity is said to result. These antagonistic peptides are said to be suitable for administration as pharmaceutical compositions to treat conditions associated with excessive levels of GH, e.g., acromegaly.
The antagonistic activity of the hGH-RH analogue "Ser.sup.9 -.psi.CH.sub.2 --NH!-Tyrl!hGH-RH(1-29)" of U.S. Pat. No. 5,084,555 was said to result from the pseudopeptide bond (i.e., a peptide bond reduced to a CH.sub.2 --NH! linkage) between the R.sup.9 and R.sup.10 residues. (It is noted that although this patent employed the seemingly redundant ".psi.CH.sub.2 --NH!" formula for the pseudo-peptide bond, actually only one such linkage had been introduced into the peptide.) However, the antagonistic properties of Ser.sup.9 -.psi.CH2--NH!-Tyr.sup.10 !hGH-RH(1-29) were said to be inferior to a conventional antagonist, N-Ac-Tyr.sup.1, D-Arg.sup.2 !GH-RH(1-29)-NH.sub.2.
U.S. Pat. No. 5,084,442 discloses cyclic lactam analogues of GH-RH which are agonists. The introduction of lactam bridge into peptides or proteins is known to reduce the number of possible conformations. Incorporation of constraints into biologically active peptides may improve receptor binding and result in analogs which have enhanced or prolonged biological activity.